Development of simultaneous analysis of tryptophan metabolites in serum and gastric juice – an investigation towards establishing a biomarker test for gastric cancer diagnosis

To evaluate changes in tryptophan metabolism and discover diagnostic biomarkers for gastric cancer, a quantitative method was developed for tryptophan and its seven metabolites (indole‐3‐lactic acid, anthranilic acid, serotonin, nicotinic acid, kynurenic acid, kynurenine and 3‐indoxyl sulfate) in both human serum and gastric juice using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Serum and gastric juice were prepared with a simple protein precipitation using aqueous 0.1% formic acid and acetonitrile. As a result, it was found that the kynurenine pathway of tryptophan metabolism was activated in gastric cancer and that the metabolic ratio of kynurenine/tryptophan, which reflects the enzyme activity of indoleamine‐2,3‐dioxygenase, was associated with the observed metabolic changes. Finally, the investigation of tryptophan metabolites, especially kynurenic acid, in serum and gastric juice might serve as biomarkers for gastric cancer. The findings in this study provide critical information of tryptophan metabolism which can be applied to a serum‐based diagnostic test for gastric cancer.     

Determination of tryptophan and its metabolites in human plasma and serum by high-performance liquid chromatography with automated sample clean-up system

An automated high-performance liquid chromatographic method that incorporates direct injection of biological samples followed by chromatographic sample clean-up in a precolumn is described for the determination of tryptophan and its metabolites in human plasma and serum. The system gave reproducible data with a coefficient of variation of less than 3% with a sample size of 100 microliters of human plasma. The major tryptophan metabolites found in 100 microliters of human plasma were kynurenine, indolelactic acid, indoleacetic acid, indolepropionic acid, serotonin and 5-hydroxyindoleacetic acid. The level of tryptophan and kynurenine in individuals was constant in comparison with other metabolites. Analysis of samples from normal controls, diabetics, gravida and their foetuses showed a tendency for tryptophan metabolites to be low in maternal plasma.     

蚕丝丝素中色氨酸含量及其在丝素纤维中的径向分布研究

通过双波长分光光度法和对二甲氨基苯甲醛显色法研究桑蚕春茧内、中、外各茧层丝素中的色氨酸含量和色氨酸在丝素纤维中的径向分布状况 ,并探讨了光、热处理后蚕丝丝素中色氨酸含量的变化 .结果表明 ,外、中、内各茧层丝素的色氨酸含量不一致 ,由外至内各茧层丝素中色氨酸含量依次降低 ;色氨酸在丝素纤维的径向分布不均一 ,呈现色氨酸在易溶落的最外表面层含量最多 ,次表层相对较少 ,中间层较多 ,而内层最少的分布状态 ;光或热处理均会破坏丝素中色氨酸 ,使色氨酸含量明显下降 .     

Chromatographic analysis of tryptophan metabolites

The kynurenine pathway generates multiple tryptophan metabolites called collectively kynurenines and leads to formation of the enzyme cofactor nicotinamide adenine dinucleotide. The first step in this pathway is tryptophan degradation, initiated by the rate‐limiting enzymes indoleamine 2,3‐dioxygenase, or tryptophan 2,3‐dioxygenase, depending on the tissue. The balanced kynurenine metabolism, which has been a subject of multiple studies in last decades, plays an important role in several physiological and pathological conditions such as infections, autoimmunity, neurological disorders, cancer, cataracts, as well as pregnancy. Understanding the regulation of tryptophan depletion provide novel diagnostic and treatment opportunities, however it requires reliable methods for quantification of kynurenines in biological samples with complex composition (body fluids, tissues, or cells). Trace concentrations, interference of sample components, and instability of some tryptophan metabolites need to be addressed using analytical methods. The novel separation approaches and optimized extraction protocols help to overcome difficulties in analyzing kynurenines within the complex tissue material. Recent developments in chromatography coupled with mass spectrometry provide new opportunity for quantification of tryptophan and its degradation products in various biological samples. In this review, we present current accomplishments in the chromatographic methodologies proposed for detection of tryptophan metabolites and provide a guide for choosing the optimal approach.     

Determination of tryptophan and kynurenine in brain microdialysis samples by capillary electrophoresis with electrochemical detection

Capillary electrophoresis with electrochemical detection (CEEC) is evaluated for the determination of tryptophan and kynurenine in microdialysis samples obtained from rat brain. These compounds were separated from all other electroactive metabolites of tryptophan. Limits of detection for both compounds were in the low attomole range. The response was linear for kynurenine between 4.9 and 980 fmol injected with a correlation coefficient of 0.9992 (n = 12). The system was evaluated for monitoring tryptophan and kynurenine in the extracellular fluid of the rat brain following systemic administration of tryptophan.     

PHOTOPHARMACOLOGY OF THE TRANQUILIZER CHLORDIAZEPOXIDE IN RELATION TO ITS PHOTOTOXICITY

Abstract— This study demonstrates that photobiological effects by exogenous molecules are not per se restricted to the skin. As an example the photopharmacology of chlordiazepoxide (e.g. Librium^R) was studied. Rats being exposed to UV-A after administration of ¹⁴C₂ labeled chlordiazepoxide (CDZ) showed a marked change in the pattern of urinary metabolites; the quantity of metabolites without N₄-oxide function increased, whereas that of compounds with N₄-oxide function decreased. Covalent binding to organ tissue of especially liver and skin of the irradiated rats was found. This would mean that in the irradiated rats oxaziridines which may also react with tissue of the inner organs are formed from CDZ and its N₄-oxymetabolites.
Further, the combination of light and CDZ leads to a 30% decrease in weight of the liver, indicating liver damage. This is also supported by the percentage of conjugated urinary metabolites being 1.5–2.5 times lower than in the non-irradiated CDZ-treated rats, possibly caused by a decrease of enzymatic activity.
The administration of CDZ-oxaziridine to non-irradiated rats provoked the same effects, such as a decrease of the weight of the liver comparable to that of irradiated CDZ-treated rats. On the other hand the combination of light and treatment with N₄-desoxy CDZ did not have any effect, as expected.     

High-voltage electrophoretic separation of tryptophan metabolites of the kynurenine pathway

An analytical separation method for tryptophan and its seven metabolites of the kynurenine pathway by high-voltage paper electrophoresis is presented. Anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid, kynurenine, nicotinic acid, quinolinic acid, xanthurenic acid and unmetabolized tryptophan are measured in urine. Using radioactive labelling and scintillation counting as a quantification method, the relative standard deviation varied from 3.5% to 14.4%, corresponding to kynurenine and nicotinic acid, respectively. The recovery of labelled tryptophan added to urine was 95%. An advantage of the electrophoretic method is the minor tailing of spots and, hence, a good resolution of the components. For the monovalent anions of the kynurenine pathway metabolites, a linear correlation (r = 0.9996) was found between the experimental relative electrophoretic mobility and the quantity M-2/3, where M is the molecular mass of the anion.     

基于HPLC/Q-TOF MS的4种农药联合暴露人群的代谢组学研究

采用基于高效液相色谱-飞行时间质谱联用(HPLC-TOF MS)的代谢组学方法,研究了啶虫脒、高效氯氟氰菊酯、联苯菊酯、甲氨基阿维菌素苯甲酸盐4种农药联合暴露所致的施药人群尿液中内源性代谢物的变化。采集30位农民喷洒4种复合农药前和喷洒农药期1,3,5,7 d的尿液进行检测。提取正常尿液中常见代谢物并通过质控样品评价手段进行分析,结果表明该方法具有良好的稳定性和精密度,可用于尿液中代谢物分析。多变量分析结果表明,暴露人群施药前后尿液的代谢物含量存在较大差异。对选取的36个差异离子进行鉴定,确定了8个生物标志物的结构。结果显示联合暴露组人群尿液中多巴胺、5-羟色胺、酪氨酸、色氨酸、牛磺酸和马尿酸的含量显著下降;犬尿素和肌酸的含量显著上升。4种农药联合暴露导致接触人群尿液中色氨酸代谢途径的中间产物含量降低,肝代谢和能量代谢相关的代谢物蓄积,可能与神经系统和肝脏功能的受损有关。     

轻稀土镧、铈急性生物效应的血清1H NMR谱代谢组学研究

采用现代核磁共振和模式识别技术,通过分析腹腔注射给药La(NO3)3(2，10和50 mg/kg体重)和Ce(NO3)3(2，10和50 mg/kg体重) 48 h后大鼠血清的核磁共振氢谱,由内源性代谢物浓度的变化研究了两种稀土化合物在大鼠体内的急性生物效应,利用主成分分析法对两者进行了分类比较. 在低剂量(2 mg/kg体重)组La(NO3)3和Ce(NO3)3血清中， 乳酸和肌酸酐等内源性代谢物浓度发生了微小变化； 在高剂量组(10和50 mg/kg体重)中， 含量变化较大的重要内源性代谢物包括3-羟丁酸、丙氨酸、 肌酸酐、 丙酮、 乙酸、 琥珀酸和葡萄糖等,其中3-羟丁酸和丙氨酸等在La的剂量组中变化较大,Ce的剂量组血清中丙酮、 琥珀酸和葡萄糖含量变化明显. 实验结果表明， 低剂量La和Ce的毒性较低,并对机体影响较小,高剂量则对大鼠肝脏造成损伤,且Ce的毒性大于La.     

Precision-cut liver slices from rats of different ages: basal cytochrome P450-dependent monooxygenase activities and inducibility

The biotransformation capacity – of the cytochrome P450 (CYP) system for example – is lower but inducibility is more pronounced in neonates than in adults. On the other hand, both enzyme activities and inducibility decline with senescence. Precision-cut rat liver slices are widely used as an in vitro tool for the examination of drug toxicity, xenobiotic metabolism or enzyme induction. The aim of the present study was to assess whether age-related changes in CYP activities and induction observed in vivo are also mirrored in vitro in liver slices. For this purpose, different CYP model reactions were measured in precision-cut liver slices from one-day-old, 40-day-old and one-year-old rats after in vitro exposure to various inducers. Similar to the in vivo situation, basal CYP activities were distinctly lower and inducibility was much more pronounced in liver slices from neonatal than in those from adult animals. Also, enzyme activities were mostly somewhat lower in liver slices from aged rats compared to those from 40-day-old rats. However, CYP inducibility was less pronounced than with younger animals too. Thus, precision-cut rat liver slices are a suitable in vitro tool for investigating age-related changes in CYP activities and induction as well as developmental differences in drug metabolism and toxicity.     

Biliary excretion of essential trace elements in rats under oxidative stress caused by selenium deficiency

The excretion of essential trace elements, namely, Se, Sr, As, Mn, Co, V, Fe, and Zn into the bile of Se-deficient (SeD) Wistar male rats was studied using the multitracer (MT) technique, and instrumental neutron activation analysis (INAA). Normal and Se-control (SeC) rat groups were used as reference groups to compare the effects of Se levels on the behaviors of the essential trace elements. The excretion (% dose) of Se, Sr, As, Mn, Co, and V increased with Se levels in the liver. The biliary excretion of Mn and As dramatically enhanced for SeC rats compared with SeD rats, while that of V accelerated a little for SeC rats. The radioactivity levels of ⁵⁹Fe and ⁶⁵Zn in the MT tracer solution were insufficient to measure their excretion into bile. The role of glutathione and bilirubin for biliary excretion of the metals was discussed in relation to Se levels in rat liver.     

THE EFFECT OF ENVIRONMENTAL LIGHT EXPOSURE ON DRUG-INDUCED PORPHYRIA IN THE RAT

Abstract— The porphyrinogenic drug, 3,5-diethoxycarbonyl 1,4-dihydrocollidine (DDC) when administered orally to rats evoked large increases in hepatic δ-aminolevulinic acid synthetase (ALAS) activity and hepatic protoporphyrin levels. These increases varied markedly according to light exposure patterns of the animals. DDC-treated animals continuously exposed for one week to fluorescent Blacklight lamps (Westinghouse FS-40) demonstrated a greater than two-fold increase in hepatic ALAS and a greater than threefold increase in liver protoporphyrin levels as compared to DDC-treated animals exposed to ambient light-dark cycling. Furthermore, the skin of porphyric animals continuously exposed to light showed larger increases in porphyrin content as compared to rats exposed to ambient light. These studies indicate that light exposure patterns can profoundly alter the activity of the hepatic heme pathway in the rat and suggest that light exposure could play a role in the production of drug-induced porphyria in man.     

EFFECT OF HEMATOPORPHYRIN DERIVATIVE ON HEMATOLOGICAL PARAMETERS IN RATS

Abstract— Wistar rats were injected with hematoporphyrin derivative (Hpd) intraperitoneally and kept in the dark. Rats were sacrificed 2, 24, 48 and 72 h after injection. It was observed that Hpd in the dark did not affect the hemoglobin content and number of erythrocytes, while the leukocyte count was increased and blood pH decreased. Blood levels of glucose and lactate were increased significantly. Because the food intake was similar in all the groups, glycogenolysis was suspected to be the source of increased glucose levels in blood. However, a significant increase in the glycogen content of the livers of Hpd-treated rats was observed, which rules out glycogenolysis. Hyperglycemia may result due to a number of reasons such as stimulation of the central nervous pathways innervating the liver and adrenal medulla, excessive glucogenesis in liver from glycogen and noncarbohydrate sources, emotional stress, anesthesia and hormonal effects. The present study rules out hyperglycemia due to anesthesia and glucogenesis in the liver. Maintenance of blood glucose levels is a highly complex mechanism. Further investigations to understand these mechanisms are in progress.     

PROFLAVINE-SENSITIZED PHOTOOXIDATION OF TRYPTOPHAN AND RELATED PEPTIDES

Abstract— The photodynamic action of proflavine on the amino acids cystine, rnethionine, tyrosine, histidine, tryptophan and related peptides was tested in anhydrous formic and acetic acids. Only rnethionine and tryptophan were found to be photooxidized: the former, whether free or bound in a polypeptide chain, is quantitatively converted to methioninesulphoxide; the latter, when free, is fragmented to several compounds, which may be divided into two main classes: melanines and kynurenine derivatives. Whereas masking the carboxyl group of tryptophan has no effect on the photooxidation products, N-substituted derivatives of tryptophan are converted in high yields to the corre-sponding kynurenine compounds. The possible applications of the method to biologically active polypeptides are discussed.     

Immunochemical detection of metabolites of parent and nitro polycyclic aromatic hydrocarbons in urine samples from persons occupationally exposed to diesel exhaust

An immunochemical assay was developed for the detection of metabolites excreted in urine as a result of occupational exposure to PAHs and nitro-PAHs. These metabolites were analyzed in a competitive ELISA, using an existing antibody (4D5) that has been developed against 6-aminobenzo[a]pyrene coupled to bovine serum albumine (B[a]P-BSA). A solid phase extraction (SPE) work-up procedure was optimized by dilution of pooled urine samples from rats exposed to 1-NP, in human urine. The application was validated by comparison of test results with urinary excreted 1-hydroxypyrene (1-OH-P) levels in a study among 28 railroad workers. Excretion of urinary metabolites was determined over two consecutive workdays. The 24 h average excretion of metabolites as determined in the immunoassay was not related to levels of particulate matter in the breathing zones of workers, not to 1-OH-P excretion levels of the day of urine collection. However, it was significantly associated with the personal dust exposure of the day before (P<0.0001), suggesting slow excretion of urinary metabolites. Smoking habits caused minor interference (P<0.1).     

硝酸镥急性毒性的体液核磁共振氢谱研究

采用现代核磁共振技术,通过分析灌胃给药0．01、0．05、0．2、2、10和100ms／kg剂量Lu(NO3)3 24h内大鼠尿液及24h后大鼠血清的核磁共振氢谱(^1HNMR),由体液中内源性代谢物浓度的变化研究了稀土化合物在动物体内急性毒性。较高剂量组体液中的氨基酸、尿囊素、柠檬酸、氮氧三甲胺和肌酸酐等重要内源性代谢物的核磁共振谱峰强度发生了明显的变化,说明动物体内的代谢物出现异常：高剂量的稀土的引入可能使动物肾脏和肝脏均受到损害,且受损程度随稀土剂量的增高而渐趋严重。     

NMR ANALYSES OF TRYPTOPHAN-8-METHOXYPSORALEN PHOTOREACTION PRODUCTS FORMED IN THE PRESENCE OF OXYGEN

Proton-made spectroscopy was performed on solutions of l -tryptophan and 8-mcthoxypsoralen (8-MOP) in either D₂O or DMSO-d₆ in the presence of oxygen before and after irradiation with 360 nm monochromatic light. Irradiation results in the loss of hydrogen atoms at the 3. 4 and 4'. 5' positions of 8-MOP and at the indole C₂ position of tryptophan. Changes in the aliphatic regions of the spectra also occur with irradiation. It is postulated that generation of photoreaction products between 8-MOP and tryptophan involves the 3.4 and 4'.5' positions of 8-MOP and the imidazole moiety of tryptophan.
Reprint requests to: Dr J. Megaw, Laboratory for Ophthalmic Research, Emory University School of Medicine, Atlanta. Georgia 30322. USA.     

Determination of Kynurenine and Tryptophan in Human Plasma by Stacking-Micellar Electrokinetic Chromatography

Kynurenine and tryptophan are important biomarkers for disorders in nervous and immune systems, while the low content of kynurenine in human plasma makes the determination by routine approaches difficult. In this work, an on-line preconcentration method for capillary electrophoresis was developed for the quantification of kynurenine and tryptophan. A concomitant sweeping phenomenon was observed due to the combination of micellar electrokinetic chromatography and stacking. The poor sensitivity in capillary electrophoresis was improved so that the determination of low concentrations of kynurenine became possible. Parameters that may affect the efficiency of the on-line concentration were systematically investigated and optimized. A large volume sample injection was employed for sampling at 100 mbar for 112 sec. A voltage of ?20 kV was applied, and the ionic sample matrix was removed with stacking while analytes were kept in the capillary. The voltage was switched to +18 kV and micellar electrokinetic chromatography was performed for separation and determination of kynurenine and tryptophan. Based on peak heights, the concentration factors for kynurenine and tryptophan were 33 and 31, respectively. The analytical performance of the established method was studied; good linearity of kynurenine was observed in the range of 0.3–30 µM (R² = 0.9988), and 0.3–60 µM (R² = 0.9963) was obtained for tryptophan. LODs of 0.15 µM for kynurenine and 0.30 µM for tryptophan were obtained. The optimum method was successfully tested in human plasma; kynurenine and tryptophan were well identified, which suggests that this method has potential for the analysis of biological samples.     

Factors affecting the ultramicro-determination of tryptophan,kynurenine and their derivatives

The factors affecting the ultramicrodetermination of tryptophan and kynurenine were investigated by paper electrophoresis, paper chromatography and spectrophotometry. The electrolyte used in electrophoresis influences the mobility, the separation and the ultraviolet spectra of tryptophan and kynurenine. The pre-treatment of filter paper for chromatography did not influence the R_F of tryptophan and kynurenine, but interfered with their quantitative determination. The effect of ultraviolet irradiation of d-, l-, and dl-tryptophan as well as d-, l-, and dl-kynurenine was studied.